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The oscillator of the cyanobacterial circadian clock relies on the ability of the KaiB protein to switch reversibly between a stable ground-state fold (gsKaiB) and an unstable fold-switched fold (fsKaiB). Rare fold-switching events by KaiB provide a critical delay in the negative feedback loop of this posttranslational oscillator. In this study, we experimentally and computationally investigate the temperature dependence of fold switching and its mechanism. We demonstrate that the stability of gsKaiB increases with temperature compared to fsKaiB and that the Q10 value for the gsKaiB → fsKaiB transition is nearly three times smaller than that for the reverse transition in a construct optimized for NMR studies. Simulations and native-state hydrogen-deuterium exchange NMR experiments suggest that fold switching can involve both partially and completely unfolded intermediates. The simulations predict that the transition state for fold switching coincides with isomerization of conserved prolines in the most rapidly exchanging region, and we confirm experimentally that proline isomerization is a rate-limiting step for fold switching. We explore the implications of our results for temperature compensation, a hallmark of circadian clocks, through a kinetic model.
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This content will become publicly available on September 17, 2026
Ultrafast photophysics of a positive reversibly switchable fluorescent protein
Kohinoor is a positive reversibly switching fluorescent protein. Ultrafast electronic and vibrational spectroscopy suggest that the chromophore switching mechanism is steered by dynamics in surrounding protein residues.
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- Award ID(s):
- 1817837
- PAR ID:
- 10650497
- Publisher / Repository:
- Royal Society of Chemistry
- Date Published:
- Journal Name:
- Chemical Science
- Volume:
- 16
- Issue:
- 36
- ISSN:
- 2041-6520
- Page Range / eLocation ID:
- 16955 to 16969
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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